An RNA pseudoknot mediates toxin translation and antitoxin inhibition

Significance Transcription–translation coupling refers to when translation initiates during mRNA transcription. While beneficial at most genes, this phenomenon constrains tight gene repression. At many type I toxin genes, cotranscriptional translation is bypassed by a two-step mechanism. First, intramolecular structure renders the nascent mRNA translationally inactive, while subsequent ribonucleolytic processing generates an active mRNA. Second, the active mRNA is silenced by an antisense small RNA (sRNA). Contrary to this established mechanism, we here suggest an alternative mechanism for bypassing cotranscriptional translation. Instead of ribonucleolytic processing, the nascent timP mRNA is activated through a structural transition, which involves the formation of a pseudoknot. The active mRNA is specifically targeted by the sRNA TimR, which destabilizes the pseudoknot to inhibit translation.


Figures S1 to S5
Tables S1 to S2 SI References

RNA extraction
Bacterial cultures were mixed with 0.2 volumes of stop solution (95% ethanol, 5% phenol) and immediately frozen in liquid nitrogen, after 30 min induction with 0.2% L-arabinose.Following centrifugation, bacterial pellet was resuspended in TE buffer (10mM Tris pH 8.0, 1mM EDTA), supplemented with 0.5 mg/ ml.1% SDS was then added in the suspension and the samples were incubated at 64 o C for 2 min.0.1 volume of sodium acetate pH 5.2 and 1 volume of acidic phenol were added in the suspension and the samples were incubated for 6 min at 64 o C, followed by centrifugation, chlorophorm extraction and ethanol precipitation.The extraxted RNA was resuspended in sterile water and stored at -20oC.The RNA integrity was determined by agarose gel electrophoresis.

Northern blot
Total RNA samples (3.5 µg) were loaded on a 6% denaturing polyacrylamide/ 8 M Urea gel electrophoresis.Radioactively labeled puC19 DNA/ MspI (HpaII) Marker (ThermoFischer) was used as a size ladder.The total RNA was transferred to a Hybond-N+ nitrocellulose membrane (Amersham, Cytiva), 0.36 A, 17 V at 4 o C for 2 hours, followed by crosslinking by UV light exposure at 1200 mJ and prehybridization with church buffer (0.5 M sodium phosphate buffer pH 7.2, 1 M EDTA, 7% SDS) for 45 min at 42 o C. Radioactively labeled  or EHO-1344 (targeting timP mRNA) was added at the hybridization buffer and incubated overnight at 42 o C. The membrane was then washed in 2x SSC/ 0.1% SDS, dried and exposed to a phosphor screen.

RNase H cleavage assay
Renatured in vitro transcribed and radioactively labeled timP mRNA (47 nM) was incubated for 10 min with EHO-2502 (6.25 µM) at 37 o C, in 1x TMN buffer.5 U RNase H (New England Biolabs) or sterile water and RNase H buffer (New England Biolabs) were added, and incubated for 12 min at 37°C.Following addition of 25 µM EDTA, the samples were phenol-chloroform extracted and precipitated with ethanol.Redissolved RNA was denatured at 95 o C for 2 min and loaded on a 6% denaturing gel at 25 V. Radioactively labeled puC19 DNA/ MspI (HpaII) Marker (ThermoFischer) was used as a size ladder.Subsequently the gel was exposed to a phosphor screen overnight.

Electromobility Shift Assays
Electromobility Shift Assays were performed in 1x EMSA buffer (25 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM MgCl2).RNA was denatured for 1 min at 95ºC in sterile water, cooled on ice for 2 min, diluted in EMSA buffer, and renatured for 5 min at 37ºC.Labeled RNA at a final concentration of 0.5 nM was mixed with 500 nM of unlabeled RNA for 20 min at 37ºC.For the time-course experiments, 0.5 nM of labeled RNA was mixed with 500 nM unlabeled RNA for 10 min in 1x EMSA buffer.Renatured RNA at 500 nM was added in the reaction mix and samples were taken in time intervals.Samples were immediately separated in running native 6% polyacrylamide gels in 0.5% TBE buffer, at 200V and 4°C.

Fig. S3 .
Fig. S3.(A) Western blot monitoring in vivo expression of timP-3xflag with 5' truncations.The truncations are indicated in Fig. S2, timP-3xflag was expressed from a plasmid for 45 min in the presence of 0.02% arabinose.GroEL served as a loading control.(B) Northern blot analysis of timP mRNA and mutants thereof expressed from an arabinose-inducible promoter on a plasmid.Probing of 5S rRNA was used as loading control.(C-D) Western blot monitoring in vivo expression of timP-3xflag stem-loop deletion (C) or point mutations (D).The mutations are indicated in Fig. S2.GroEL served as a loading control.(E) Formation of a heteroduplex between an antisense oligo and the SL3 loop was tested by RNase H cleavage for wt timP, timP-M2' or timP-M2+M2'.

Fig. S4 .
Fig. S4.(A) The same Electromobility Shift Assay as shown in Figure 4A including extended time points.Radioactive labeled TimR was incubated with an excess of unlabeled timP mRNA.(B) Northern blot analysis of the timP SD::cspE SD mRNA, and mutants thereof, expressed from an arabinose-inducible promoter on a plasmid.Probing of 5S rRNA was used as loading control.(C) Electromobility Shift Assay where radioactively labeled TimR pre-bound to unlabeled timP was challenged with an excess of unlabeled TimR or a binding-incompetent mutant (M6 TimR) after which samples were collected in time intervals.

Fig. S5 .
Fig. S5.Model for post-transcriptional regulation of the timPR.Transcription produces a translationally inactive timP mRNA, which subsequently becomes translationally active through a structural transition involving formation of a pseudoknot.TimR targets the translationally active structure, which destabilizes the pseudoknot and inhibits timP translation.

Table S1 .
Plasmids used in the study.

Table S2 .
Oligonucleotides used in the study.